Department of Physiology and Cell Biology.
Department of Internal Medicine.
Am J Respir Cell Mol Biol. 2020 Oct;63(4):519-530. doi: 10.1165/rcmb.2019-0435OC.
KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.
KD025 是一种 ROCK2 抑制剂，目前正在临床试验中用于治疗纤维化肺部疾病。KD025 的治疗效果部分归因于其对促纤维化途径和脂肪代谢的抑制。然而，尽管有证据表明肺泡-毛细血管膜破裂是纤维化肺部疾病死亡的主要原因，但 KD025 是否影响肺微血管内皮细胞（PMVEC）功能尚不清楚。我们假设 KD025 调节 PMVEC 代谢、pH 值、迁移和存活，这一系列相互关联的功能特征决定了肺屏障的完整性。我们使用从 Sprague Dawley 大鼠中分离的 PMVEC。KD025 剂量依赖性地降低乳酸生成和葡萄糖消耗。与其他代谢调节剂相比，KD025 的抑制作用更强，包括 2-脱氧葡萄糖、细胞外酸中毒、二氯乙酸和 remogliflozin。有趣的是，KD025 增加了氧化磷酸化，而 2-脱氧葡萄糖则没有。KD025 还降低了细胞内 pH 值，并诱导阴离子交换蛋白 2 的代偿性增加。KD025 抑制 PMVEC 迁移，但 fasudil（非特异性 ROCK 抑制剂）没有。我们使用 Evans Blue 染料在博来霉素肺纤维化模型中测试内皮通透性。KD025 处理的动物在不接受博来霉素治疗的情况下，基线通透性降低。在缺氧条件下，KD025 增加了 PMVEC 坏死，表现为乳酸脱氢酶释放和碘化丙啶摄取增加以及 ATP 减少；它不影响 Annexin V 结合。ROCK2 敲低对 PMVEC 代谢、pH 值和迁移没有影响，但增加了非凋亡性 caspase-3 活性。总之，我们报告 KD025 促进氧化磷酸化；降低糖酵解、细胞内 pH 值和迁移；并以 ROCK2 非依赖性方式增强肺屏障完整性。
